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Abstract:

This invention provides methods of measuring the viability of cultured
cells by detecting one or more cell death-stable proteins or enzyme
activities. Methods provided by the invention correlate viability to
relative levels of enzyme activity in cell-containing and
non-cell-containing fractions of a cell culture.

Claims:

1. A method of measuring the fraction of viable cells in a cell
population maintained in a cell culture medium comprising: (a) detecting
a cell death-stable enzyme activity in a portion of a cell culture
conditioned medium not containing cells of the cell population; (b)
detecting a cell death-stable enzyme activity in the cells and
conditioned medium of a portion of the cell culture medium containing
cells of the cell population; and (c) comparing the level of cell
death-stable enzyme activity in the cells and conditioned medium portion
of the cell culture medium containing cells to the level of cell
death-stable enzyme activity in the portion of the cell culture
conditioned medium not containing cells, wherein the cell death-stable
enzyme activity is measured by the steps comprising: (i) contacting a
sample with a substrate of the cell death-stable enzyme activity wherein
the substrate in conjugated to a detectable leaving group; and (ii)
detecting the leaving group, wherein the amount of leaving group detected
is proportional to the level of cell death-stable enzyme activity,
wherein the method additionally comprises adding an agent which modulates
the signal of the detectable leaving group, and wherein the fraction of
viable cells in the cell population is directly proportional to the
difference between the level of cell death-stable enzyme activity in the
portion of the cell culture medium containing cells and conditioned
medium and the level of cell death-stable enzyme activity in the portion
of the cell culture conditioned medium not containing cells.

2. A method of measuring the fraction of viable cells in a
tissue-engineered product maintained in a cell culture medium comprising:
(a) detecting a cell death-stable enzyme activity in a portion of a cell
culture conditioned medium not containing cells of the tissue-engineered
product; (b) detecting a cell death-stable enzyme activity in the cells
and conditioned medium of a portion of the cell culture medium containing
cells of the tissue-engineered product; and (c) comparing the level of
cell death-stable enzyme activity in the portion of the cell culture
medium containing cells and conditioned medium of the tissue-engineered
product to the level of cell death-stable enzyme activity in the portion
of the cell culture conditioned medium not containing cells of the
tissue-engineered product, wherein the tissue-engineered product
comprises a three-dimensional scaffold or matrix and cells are present in
the scaffold or matrix, and wherein the fraction of viable cells in the
tissue-engineered product is directly proportional to the difference
between the level of cell death-stable enzyme activity in the portion of
the cell culture medium containing cells and conditioned medium of the
tissue-engineered product and the level of cell death-stable enzyme
activity in the portion of the cell culture conditioned medium not
containing cells of the tissue-engineered product.

3. A method of measuring the fraction of viable cells in a
tissue-engineered product maintained in a cell culture medium comprising:
(a) providing a sample portion of the tissue engineered product
containing cells and a proportional amount of a cell culture conditioned
medium; (b) detecting a cell death-stable enzyme activity in a portion of
a cell culture conditioned medium not containing cells of the
tissue-engineered product from the sample portion provided in step (a);
(c) disrupting the membrane integrity of the cells in the sample portion
of the tissue engineered product provided in step (a); (d) detecting a
cell death-stable enzyme activity in the cells and conditioned medium of
the sample portion of the tissue-engineered product provided in step (a);
and (e) comparing the level of cell death-stable enzyme activity detected
in steps (b) and (d), wherein the tissue-engineered product comprises a
three-dimensional scaffold or matrix and cells are present in the
scaffold or matrix, and wherein the fraction of viable cells in the
tissue-engineered product is directly proportional to the difference
between the level of cell death-stable enzyme activity detected in (d)
and (b).

5. The method of claim 4, wherein the membrane integrity of the tissue
engineered product is disrupted by the addition of an amphiphile.

6. The method of claim 5, wherein the amphiphile is saponin.

7. A method of measuring the fraction of viable cells in a
tissue-engineered product maintained in a cell culture medium comprising:
(a) providing a portion of the tissue engineered product containing cells
and a proportional amount of the cell culture conditioned medium; (b)
providing a portion of the cell culture conditioned medium, provided in
step a), not containing cells of the tissue engineered product; (c)
adding saponin, and bis-(Ala-Ala-Phe)-Rhodamine-110 to the samples
provided in steps a) and b); and (d) detecting fluorescences
corresponding to cleaved bis-(Ala-Ala-Phe)-Rhodamine-110 in the samples
provided in steps a) and b), wherein the cells are human chondrocytes,
maintained in a matrix at a density of between 1.5.times.10.sup.4 and
6.times.10.sup.6 cells/cm2, and the fraction of viable cells in the
tissue engineered product is proportional to the difference divided by
the sum, of the fluorescences detected in d).

8. A method of measuring the cytotoxicity of a treatment to cells in a
tissue-engineered product maintained in a cell culture medium comprising:
(a) applying the treatment to the tissue engineered product; (b)
detecting a cell death-stable enzyme activity in a portion of a cell
culture conditioned medium not containing cells of the tissue-engineered
product; (c) detecting a cell death-stable enzyme activity in the cells
and conditioned medium of a portion of the cell culture medium containing
cells of the tissue-engineered product; and (d) comparing the level of
cell death-stable enzyme activity in the portion of the cell culture
medium containing cells of the tissue-engineered product to the level of
cell death-stable enzyme activity in the portion of the cell culture
conditioned medium not containing cells of the tissue-engineered product,
wherein the tissue-engineered product comprises a three-dimensional
scaffold or three-dimensional matrix and cells are present in the
three-dimensional scaffold or three-dimensional matrix, and wherein the
cytotoxicity of the treatment is proportional to the difference in the
fraction of viable cells in the treated tissue engineered product to an
untreated tissue engineered product, and wherein the fraction of viable
cells in the tissue-engineered product is directly proportional to the
difference between the level of cell death-stable enzyme activity in the
portion of the cell culture medium containing cells of the
tissue-engineered product and the level of cell death-stable enzyme
activity in the portion of the cell culture conditioned medium not
containing cells of the tissue-engineered product.

9. The method of claim 1, wherein the cells are mammalian.

10. The method of claim 9, wherein the cells are human.

11. The method of claim 10, wherein the cells are chondrocytes.

12. The method of claim 9, wherein the cells are grown on a matrix.

13. The method of claim 9, wherein the cells are present at a density of
between 1.5.times.10.sup.4 and 6.times.10.sup.6 cells/cm.sup.2.

14. The method of claim 1, wherein the cells are present at a high
density of at least 2.0.times.10.sup.5 cells/cm.sup.2.

15. The method of claim 14, wherein the cells are present at between
2.times.10.sup.5 and 6.times.10.sup.6 cells/cm.sup.2.

16. The method of claim 3, wherein the cell death-stable enzyme activity
is measured by the steps comprising: (a) contacting a sample with a
substrate of the cell death-stable enzyme activity wherein the substrate
in conjugated to a detectable leaving group; and (b) detection of the
leaving group, wherein the amount of leaving group detected is
proportional to the level of cell death-stable enzyme activity.

17. The method of claim 1, wherein the leaving group is chromogenic,
luminogenic, or fluorescent.

18. The method of claim 17, wherein the leaving group is fluorescent.

19. The method of claim 18, wherein the leaving group is Rhodamine-110.

20. The method of claim 16 wherein the substrate is
bis-(Ala-Ala-Phe)-Rhodamine-110.

21. The method of claim 18, wherein the leaving group is a coumarin
derivative.

22. The method of claim 21, wherein the leaving group is AMC.

23. The method of claim 22, wherein the substrate is Ala-Ala-Phe-AMC.

24. The method of claim 16, further comprising the step of adding an
agent which modulates the signal of the detectable leaving group.

25. The method of claim 1, wherein the agent which modulates the signal
of the leaving group, attenuates the signal of the leaving group.

26. The method of claim 25, wherein the agent which attenuates the signal
of the leaving group is phenol red.

27. The method of claim 26, wherein the phenol red is present at a
concentration of up to 500 mg/L.

Description:

[0001] This is a continuation of application Ser. No. 12/266,246, filed
Nov. 6, 2008, which claims the benefit of provisional application No.
60/986,751, filed Nov. 9, 2007, all of which are incorporated by
reference in its entirety.

[0002] This invention relates to the field of cell biology as well as cell
culture and tissue engineering. More specifically, the invention relates
to methods of measuring the viability of cultured cells by detecting a
protein or enzyme activity.

[0003] The field of tissue engineering (reviewed in Langer and Vacanti,
Science, 260:920-926 (1993)) centers around the use of matrices or
scaffolds to support the growth and maintenance of cells. For example,
matrix-induced autologous chondrocyte implantation (MACI® implants)
is a second-generation autologous chondrocyte implantation (ACI)
procedure used to repair catilage. In a MACI® implant,
culture-expanded chondrocytes are seeded onto a collagen-based membrane
matrix, which later facilitates surgical implantation. MACI® implants
may be used to treat cartilage defects arthroscopically or through
minimally invasive surgery.

[0004] The use of matrices in tissue engineered products presents
significant challenges to investigators trying to measure the viability
of the engineered products' constituent cells. Existing methods of
measuring cell viability rely on at least one of two features of viable
cells: the presence of an intact plasma membrane and/or their metabolic
activity. In vitro, cell death is accompanied by the loss of plasma
membrane integrity. This phenomenon can be readily observed under a
microscope using vital dyes. In the most common vital dye assay, the dye
trypan blue is added to a suspension of cells. The dye is excluded from
viable cells with an intact membrane but stains dead or dying cells with
a disrupted membrane. Alternatively, cell viability may be assessed by
measuring one or more markers of the cell's metabolic activity. One such
approach is to quantify key metabolites (e.g., ATP, NADH), which are
present in viable cells but depleted or absent from dead cells. A
complementary approach is to assay for specific enzyme activities
released from membrane-compromised cells. For example, the Cytox-Fluor
cytotoxicity assay (Promega, Madison, Wis., Cat. No. G9260) detects
proteases such as tripeptidyl peptidase released from dead cells using
the internally quenched fluorogenic peptide substrate
bis-(Ala-Ala-Phe)-Rhodamine-110.

[0005] When applied to tissue engineered products, most existing cell
viability assays require the cells to be isolated (recovered) before
assay. The isolation process, however, is often complicated, sometimes
harsh, and never 100% efficient. Measurement artifacts may arise as
viable cells are lost or killed, or as dead cells are lost, during the
procedure. For example, when evaluated by trypan blue exclusion,
recovered cells always have a near 100% viability, which fails to reflect
the true viability of the original sample from which they were obtained.
Attempts to use metabolic activity-based viability assays without first
recovering the cells are similarly unsuccessful due to matrix
interference, non-specific binding, low upper limit of detection,
inadequate range, or poor precision in different media types.
Furthermore, existing metabolic activity-based cell viability assays all
share a fundamental disadvantage, i.e., the requirement for a positive
and/or negative control, with known cell number and viability, in order
to measure the viability of the test sample. To make a valid comparison,
the cells used in the control and test samples have to be the same type
of cells from the same donor or strain, and must also have the same
metabolic profile. This approach is not applicable to tissue engineering
products where cells, seeded in 3-dimensional matrices, often acquire a
very different metabolic profile than the same cells grown in suspension
or on a 2-dimensional surface. Additionally, it is often impractical to
obtain extra cells and prepare appropriate controls in industrial
manufacturing practice where a large number of lots are assayed on a
daily basis for viability and quality control.

[0006] The viability of the cells being transplanted remains a major
determinant in successful treatment with tissue engineered products. In
light of the shortcomings of existing viability assays, there is a need
for easy, rapid, and accurate methods to measure the viability of cells
in tissue engineered products. Such methods must operate over a wide
range of cell densities, and with many different cell types, media, and
matrices. Furthermore, such cell viability methods advantageously operate
without the need for a control cell population.

[0007] The present invention provides methods to easily, rapidly, and
accurately measure the viability of cells under a variety of conditions
and without the need for control cells. The methods are based, in part,
on the discovery that the viability of cells in a cultured tissue
engineered product can be determined by detecting the fraction of one or
more enzyme activities of the cultured cells that are present in the
culture's supernatant.

[0008] It is theorized, but not relied upon for the purposes of this
invention, that upon the loss of membrane integrity which accompanies
cell death, the contents of the cell normally bound by the plasma
membrane become detectable in the cell culture supernatant. The methods
of the invention rely on the detection of a cell death-stable protein or
enzyme, i.e.; a protein or enzyme which can be detected whether it is
present in live or dead cells. The cell viability of the culture can then
be determined by detecting the relative amount of the cell death-stable
protein or enzyme in the non-cell-containing conditioned medium (e.g.
supernatant, or supporting matrix or scaffold) and the cell-containing
conditioned medium (e.g. the membrane-intact cells and associated
conditioned medium). The amount of cell death-stable protein or enzyme in
only the cells of the cell-containing conditioned medium can be
determined by disrupting the membrane integrity of membrane-intact cells,
e.g., by partial or complete lysis, measuring the total enzyme activity
in the cell-containing portion (i.e., disrupted cells and associated
conditioned medium) and then subtracting any enzyme activity contributed
by the associated conditioned medium. The value that is subtracted is
measured by assaying cell-free conditioned medium.

[0009] One aspect of the invention provides methods for measuring the
fraction of viable cells in a cell population maintained in a culture
medium by detecting a cell death-stable protein or enzyme activity in a
portion of the conditioned medium not containing cells (e.g. conditioned
medium), detecting a cell death-stable protein or enzyme activity in a
portion of the medium containing cells (e.g. cells and conditioned
medium), and comparing the level of cell death-stable protein or enzyme
activity in the two portions. By this method, the fraction of viable
cells in the population is proportional to the difference between the
level of cell death-stable protein or enzyme activity in the portion
containing cells and that of the portion not containing cells.

[0010] In some embodiments, the cells assayed may be grown on a
traditional two-dimensional cell culture substrate, e.g., glass or tissue
culture plastic. In other embodiments, the cells are supported on or in a
three-dimensional scaffold or matrix, i.e., the cells are part of a
tissue engineered product. In certain embodiments, the cells are grown on
a porcine collagen-derived matrix.

[0011] In certain embodiments, the methods of the invention further
include a step of providing a sample portion of the cell population and a
proportional amount of the non-cell containing culture conditioned
medium. The sample portion is then divided into a cell-containing (i.e.,
cells plus conditioned medium) and non-cell-containing (conditioned
medium only) fraction and processed according to the methods of the
invention.

[0012] In certain embodiments, the membrane integrity of the cells of the
sample portion is disrupted by, e.g., shearing, sonnication, low
barometric pressure, high temperature, low temperature, chemical or
enzymatic lysis, or membrane decoupling agents. In some embodiments, the
membrane integrity may be disrupted by the addition of an amphiphilic
molecule. In certain embodiments, the amphiphile is saponin.

[0013] Further aspects of the invention provide methods for measuring the
fraction of viable human chondrocytes present in the matrix of a
tissue-engineered product having a cell density of between
1.5×104 and 6×106 cells/cm2 maintained in a
culture medium. The steps include providing a portion of the tissue
engineered product which contains cells and a proportional amount of the
culture conditioned medium, providing a portion of the culture
conditioned medium not containing cells of the tissue engineered product,
adding saponin, and bis-(Ala-Ala-Phe)-Rhodamine-110 to the portions, and
detecting fluorescent signals from cleaved
bis-(Ala-Ala-Phe)-Rhodamine-110 in the two portions. In some embodiments,
Ala-Ala-Phe-AMC, or another substrate with a conjugated leaving group can
replace bis-(Ala-Ala-Phe)-Rhodamine-110 in these methods. The fraction of
viable cells in the tissue engineered product is proportional to the
difference in fluorescent signal strength between the cell-containing and
non-cell-containing portions divided by the total amount of the
fluorescent signal in both portions.

[0014] In another aspect, the invention provides methods of determining
the cytotoxicity of a test treatment (e.g., treatment with
pharmacological, or biological compounds; or exposure to various
conditions, e.g., of osmolarity, pH, temperature, or barometric pressure;
or photic, electric or mechanical treatments; or combinations of these)
to a test population of cultured cells. The method entails applying the
test treatment to the test population, measuring the fraction of viable
cells in the test population by the methods of the invention, and
comparing the measured viability of the test population to the viability
of an untreated population ("Control population") of the cultured cells.

[0015] The methods of the invention may be used with a variety of cells
under a variety of conditions. In some embodiments, the cells may be
mammalian (e.g., human, primate, ovine, bovine, porcine, equine, feline,
canine, or rodent). In certain embodiments, the cells are human. Cells
derived from any source tissue may be used in the methods of the
invention. In particular embodiments the cells are chondrocytes.

[0016] The methods of the invention may be used with cells at a wide range
of densities. In some embodiments the cells are present at a density of
between 1.5×104 and 6×106 cells/cm2. In other
embodiments, the cells may be present at a density of between
2.2×104 and 2.8×106 cells/cm2, between
3.5×104 and 2.8×106, or between 5×104
and 1×106 cells/cm2. In certain embodiments, the cells
may be present at a high density of at least 2.0×105,
5.0×105, 1.0×106, 2.0×106,
2.8×106, 3×106, 4×106, 5×106,
6×106, 8×106, 10×106 cells/cm2, or
still higher densities.

[0017] In some embodiments of the invention, the cell death-stable enzyme
activity is measured by contacting a sample portion with a substrate of
the cell death-stable enzyme activity, where the substrate is conjugated
to a detectable leaving group, and then detecting the leaving group. By
this method, the amount of leaving group detected is proportional to the
level of cell death-stable enzyme activity present in the sample portion.
In various embodiments, the leaving group may be chromogenic,
luminogenic, or fluorogenic. In particular embodiments, the leaving group
is fluorescent. In certain embodiments, the leaving group is
Rhodamine-110. In other embodiments, the leaving group is a coumarin
derivative, e.g., 7-amino-4-methyl coumarin (AMC).

[0018] A substrate for an enzyme activity can be any molecule processable
by the enzyme. In certain embodiments, the substrate is a tripeptide. In
some embodiments, the substrate is bis-(Ala-Ala-Phe)-Rhodamine-110. In
other embodiments, the substrate is Ala-Ala-Phe-AMC.

[0019] In some embodiments, the methods of the invention may further
include the step of adding an agent which modulates (e.g.
enhances/increases or attenuates/decreases) the signal of a leaving
group. In some embodiments, the agent may modulate the signal by at least
5, 10, 15, 20, 40, 60, or 80%; or more than 1, 2, 3, 5, 10, 50, or
100-fold. In certain embodiments, the agent which modulates the signal of
the leaving group, acts by attenuating the signal of the leaving group.
In particular embodiments, the agent that attenuates the signal of the
leaving group is phenol red. In some embodiments, the phenol red may be
present at a concentration of up to 10, 20, 40, 60, 70, 100, 150, 200
mg/L, or more.

[0020] In various embodiments of the invention, the cell death-stable
enzyme activity detected may be, e.g., anabolic or catabolic, an
oxidoreductase, transferase, hydrolase, lyase, kinase, phosphatase,
isomerase, or ligase. In some embodiments the cell death-stable enzyme
activity may be proteolytic, e.g., one or more tripeptidyl peptidases,
chymotrypsin, or chymotrypsin-like enzymes, such as calpain.

[0021] In some embodiments, the cell death-stable protein or enzyme
activity is a protein or enzyme activity that is stable following either
necrotic, programmed cell death, or both (and preferably stable following
either form of cell death). In other embodiments, the cell death-stable
protein or enzyme activity is a necrotically stable protein or enzyme
activity. In still other embodiments, the cell death-stable protein or
enzyme activity is a programmed cell death-stable protein or enzyme
activity.

[0022] In some embodiments, the methods of the invention can include a
quality control assay. In such embodiments, the methods of the invention
may further include the step of detecting a contaminant-specific enzyme
activity in either the cell containing or non-cell-containing portions,
or both. Detecting a contaminant-specific enzyme activity is indicative
of culture contamination.

[0023] The accompanying drawings, which are incorporated in and constitute
a part of this specification, illustrate several embodiments of the
invention and together with the description, further serve to explain the
principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 is a schematic depiction of a cell viability assay.

[0025] FIG. 2A is a graphical representation of an experiment which
demonstrates that the enzyme activity present in the cells and
supernatant of a sample is linearly related to the density of the cell
population.

[0026] FIG. 2B is a graphical representation of an experiment which
demonstrates that the enzyme activity present in the supernatant of a
sample is linearly related to the density of the cell population.

[0027] FIG. 3 is a graphical representation of an experiment which
demonstrates that the disclosed assay accurately predicts viability.

[0028] FIG. 4 is a graphical representation of an experiment which
demonstrates that the addition of phenol red does not affect the accuracy
of the disclosed assay.

[0029] FIG. 5 is a graphical representation of an experiment which
demonstrates that the accuracy of the disclosed assay is unaffected by
use of an alternative matrix.

[0030] FIG. 6 is a graphical representation of an experiment which
demonstrates that the accuracy of the disclosed assay is unaffected by
the absence of a matrix.

[0031] FIG. 7 is a graphical representation of an experiment which
demonstrates that the accuracy of the disclosed assay is unaffected by
the use of non-human cells.

[0032] FIG. 8 is a graphical representation of an experiment which
demonstrates that the disclosed assay can use substrates with leaving
groups other than Rhodamine.

[0035] "Surface area" as used herein, e.g., square area, cm2, refers
to the macroscopic surface area of a substrate, i.e., the Z axis
projection of the surface onto the two dimensional plane.

[0036] "Density" as used herein, means an average number of some
substance, e.g., a cell or other object, per unit area or volume. Most
frequently in this application, density will be in the context of a cell
density: the number of cells per unit of surface area. This average
quantity is approximated by dividing the number of cells seeded by the
macroscopic surface area of the surface on which they are grown. This
definition contemplates both two-dimensional surfaces, as well as
three-dimensional structures or lattices.

[0037] The term "medium" as used in this application, refers to all
components which support the growth or maintenance of cells in culture.
This may include traditional liquid cell culture medium and any
additional factors that said medium may contain. These factors may
include, for example, serum, antibiotics, growth factors, pharmacological
agents, buffers, pH indicators, and the like. Medium shall not generally
refer to any matrix or support upon, or within, which the cells are
maintained or grown unless clearly indicated otherwise. Accordingly, in a
tissue engineered product, the matrix is typically part of the
cell-containing portion.

[0038] Accordingly, "a portion of the cell culture medium not containing
cells" includes a liquid portion of the medium, and not any
cell-containing matrix. Similarly, "a portion of the cell culture medium
containing cells" includes either isolated cells or matrix-associated
cells, in association with medium.

[0039] By "conditioned medium" it is meant medium which has been contacted
with cells to allow for the composition of the medium to be modified,
e.g. by the uptake or release of one or more metabolites, nutrients, or
factors, e.g., one or more cell death-stable proteins or enzyme
activities. Unless otherwise indicated, conditioned medium generally
means medium which has been in contact with a cell population so as to
collect cell death-stable protein or enzyme activity from cells with
compromised membrane integrity.

[0040] As used in this application, "detectable leaving group" refers to a
product of an enzymatic reaction that may be used to monitor the progress
of an enzymatic reaction.

[0041] By "proteome" it is meant the set of all proteins expressed in a
group of cells.

[0042] "Recombinant" herein refers to non-native biological molecules,
e.g., nucleic acids, their transcriptional or translational products, or
cells containing any of the above.

[0043] By "intrinsic activity" of an enzyme it is meant its Vmax;
i.e., the rate of product production when only the enzyme's ability to
process substrate is limiting; reaction conditions are otherwise
optimized for the enzyme activity.

[0044] "Membrane-intact" as used in this application means the ability to
exclude the dye trypan blue under standard laboratory conditions.

[0045] By "scaling factor" it is meant a numerical constant determined for
a particular assay condition.

[0046] "Relative measure" in this application refers to expressing a
quantity as a function of a reference value; e.g., expressing one value
as a fraction of another.

[0047] "Absolute measure" in this application means the actual numerical
value of some quantity, i.e., not a relative measure.

EXEMPLARY EMBODIMENTS

[0048] The invention provides methods to calculate viability based upon
the relative measure of a cell death-stable protein or enzyme activity in
two fractions of a sample. Unlike vital dye assays, there is no need to
recover cells from the matrix. Accordingly, the methods of the invention
may eliminate the measurement artifacts associated with prior methods,
e.g., losing cells during the process, underestimating or overestimating
viability.

Determining Cell Viability

[0049] The invention provides methods of measuring the fraction of viable
cells in a population maintained in a culture medium. This is done by
comparing the amount of a cell death-stable enzyme activity in a portion
of the conditioned medium not containing cells to the amount of the cell
death-stable protein or enzyme in a portion of the conditioned medium
containing cells. In general, the methods of the invention comprise three
steps:

[0050] (1) a sample is divided into two portions, a cell-containing
portion ("X") and a non-cell-containing portion ("Y");

[0051] (2) the cells of the cell-containing portion (or a sample taken
from the cell-containing portion) are lysed; and

[0052] (3) the amount of the cell death-stable protein or enzyme in each
portion ("X" and "Y") is detected or measured. The skilled artisan can
convert these measurements to the fraction of viable cells in the cell
population in a variety of ways.

[0053] In one example, the conditioned medium is first divided equally
into two portions (i.e., halves), a cell-containing portion ("X") and a
non-cell containing portion ("Y"). As shown in FIG. 1 and described in
Example 1, when the cell-containing portion ("Y") has half of the
conditioned medium of the sample, and the remaining half is in the
non-cell-containing portion ("X"), then the fractional viability is
simply the difference divided by the sum of the activities, i.e.,

The numerator of this expression is the enzyme activity present in the
membrane-intact cells (the amount in the cell-containing portion, i.e.
cells and conditioned medium, less the amount present in the conditioned
medium alone), while the denominator is the total amount of enzyme
activity present in the sample. If the measured enzyme activity present
in the cell containing and non-cell-containing portions of the sample are
500 and 50, then the fractional viability is

500 - 50 500 + 50 = 450 550 ≈ 0.82 . ##EQU00002##

[0054] Of course, in the first step of the methods of the invention, the
conditioned medium in the sample need not be divided equally between the
cell-containing and non-cell containing portions. Where the fraction of
the total conditioned medium in the sample is not divided equally between
the two portions, the fractional viability is then given by

X - cY X + Y ##EQU00003## where c = 1 - f f .
##EQU00003.2##

In the first expression, c is a scaling factor which adjusts for the
volume of conditioned medium in the cell free portion assayed, relative
to the volume of conditioned medium in the cell containing portion
assayed. This scaling factor is a function of the non-zero, decimal
fraction f of the total sample conditioned medium present in portion Y,
the sample portion not containing cells. In an illustrative example, a
sample of a tissue engineered product is divided into:

[0055] (1) a portion X, containing the cells and 25% of the sample
conditioned medium; and

[0058] The foregoing discussion included detailed means to calculate
viability using methods provided by the invention. This may be in the
context of a culture grown solely for assay, or for the purpose of
estimating the viability of some larger population. For example, in a
"lot release" assay for a tissue engineered product, the viability of the
product's constituent cells are determined by taking a sample of the
product (i.e., a biopsy), and some of the overlying conditioned medium.
In its simplest form, the percentage of the total conditioned medium
overlying the product and the percentage of the product's total surface
area or volume (and therefore cells) biopsied, are the same. For example,
if a biopsy includes about 2% of the cells of the product, about 2% of
the volume of conditioned medium overlying the product should also be
present in the sample. Sampling may be done by taking either the cells
and conditioned medium together, or in series (in either order), or some
combination of the two techniques (e.g., take cells and some conditioned
medium, then remove additional conditioned medium) using any number of
steps contemplated by the skilled artisan. The equations above implicitly
assume this 1:1 percent cell to percent volume ratio in the sample.

[0059] The skilled practitioner will appreciate that the 1:1 cell to
volume sampling ratio may be varied, and that particular cell types,
products, or culture conditions may be amenable to, or even require,
altered ratios of cells and conditioned medium in a sample. As would be
apparent to the skilled artisan, particular modifications to the
calculations presented above should be employed depending on the sampling
strategy used. For example, the ratio of percent conditioned medium
volume to percent cells in a sample deviates from 1, then the viability
of the sample can

where α=the ratio of percent of the total cells to percent of the
total conditioned medium volume in the sample. Thus, if a sample includes
2% of the total cells and 4% of the total volume of conditioned medium
(i.e., the ratio of the percentage of total cells and percent of
conditioned medium is less than 1), and the activity in the "X" and "Y"
sample portions are 1000 and 200, respectively, then

Alternatively, the ratio of percent cells to percent conditioned medium
volume to in a sample may greater than 1. Thus, if a sample includes 5%
of the total cells and 1% of the total conditioned medium, and the
activity in the "X" and "Y" sample portions are 820 and 20, respectively,
then

viability = 820 - 20 820 + 20 + 2 × 20 × ( 5 - 1 )
= 0.8 ##EQU00008##

Notably, in these examples, the correction only needed to be made in the
denominators of the equations already presented above. This revised
equation assumes that the total volume of conditioned medium present in
the sample is divided equally between the "X" and "Y" portions assayed.
When the conditioned medium is not divided equally, this same denominator
correction may be applied to the equation, already provided, for
situations where the conditioned medium is not divided equally between
the "X" and "Y" portions of a sample.

[0060] The methods for determining cell viability provided by the
invention eliminate the need for control cells. Control cells are used to
calibrate an enzyme-based assay for a particular culture medium, matrix,
or cell type. This is, in part, because existing assays make absolute
measures of protein or enzyme activity. Absolute measures of protein or
enzyme activity can be affected by the presence or absence of, for
example, serum, supplements, vitamins, phenol red, or matrix/substrate.
In addition, absolute measures of protein or enzyme activity can be
affected by intrinsic donor-to-donor, strain-to-strain, and cell
passage-to-passage variabilities.

[0061] Additionally, existing methods often saturate at even the low end
of densities used in applications such as tissue engineering. The methods
provided by the invention are useful for measuring viability in high cell
density applications, such as tissue engineering.

[0062] A cell death-stable protein or enzyme activity is one that persists
at detectable levels through cell death occurring by various mechanisms,
e.g., programmed cell death (an energy-requiring process) or necrosis (a
non-energy-requiring process). Because various cell death processes
affect different proteins to different extents, a cell death-stable
protein may be programmed cell death-stable, necrotically stable, or
both. For an overview of cell death, see, e.g., Guimaraes and Linden,
Eur. J. Biochem., 271:1638-1650 (2004) and Hengartner, Nature, 407:770-6
(2000).

[0063] In some embodiments, the relative concentration of the cell
death-stable protein or enzyme activity is unaffected, or changes no more
than 5, 10, 15, 20, 40, 60, or 80%, or no more than 1, 2, or 3 fold in
cells having undergone a cell death process, relative to cells that have
not undergone the cell death process. In certain embodiments, the
half-life of a cell death-stable protein or enzyme activity may be about
30, 60, 90, or 120 minutes; about 2, 3, 4, 5, 6, 8, 10, or 12 hours; or
up to about 1, 2, 3, 4 days, or more.

[0064] The skilled artisan will appreciate that proteins or enzyme
activities that may be suitable for the present invention can be
identified by various means. For example, analysis of the gene expression
profile of cells undergoing a cell death process, relative to that of
cells not undergoing a cell death process, can be used to identify genes
whose protein products are cell death-stable proteins or enzyme
activities. Such genes may be differentially expressed no more than 5,
10, 15, 20, 40, 60, or 80%, or no more than 1, 2, or 3 fold in cells
undergoing a cell death process, relative to cells that are not
undergoing a cell death process. The skilled artisan will recognize that
genes identified this way must be further evaluated for the stability of
the protein product or enzyme activity under different cell death
processes.

[0065] Cell death-stable proteins or enzyme activities should be confined
by the periphery of the cell, e.g., on or within the plasma membrane, in
the cytosol, or within a membrane-bound organelle. Target molecules
should not be secreted proteins because the origin of such a protein or
enzyme activity, i.e. whether from viable or non-viable cells, cannot be
readily determined. The cell death-stable protein or enzyme activity, if
confined within the plasma membrane, must become assayable upon loss of
membrane integrity.

[0066] The membrane integrity of the cells in the cell population may be
disrupted by a variety of means known to the skilled artisan. Such means
should preserve all or most of the cell death-stable protein or enzyme
activity. For example, cell membrane integrity may be disrupted by
shearing, sonication, vacuum, high temperature, low temperature (e.g.,
freezing), chemical or enzymatic lysis, or membrane decoupling agents.
Chemical lysis may be achieved by incubation with amphiphilic molecules
such as soaps, detergents, or certain glycosides (e.g., saponin). The
amount of chemical lysis agent may be adjusted to achieve the desired
effect. For example, saponin may be used at a final concentration of
between 0.01% and 2% (WN), e.g., between 0.05% and 0.5%.

[0067] The cell death-stable protein or enzyme activity may be either a
naturally occurring component of the cell population's proteome, or a
non-naturally occurring component, e.g., an expressed recombinant
protein(s) or enzyme activity. Such recombinant molecules may be
introduced by routine methods known in the art, and may be stably or
transiently expressed, i.e., integrated into the genome, or plasmid
based. See, e.g., Joseph Sambrook and David Russell, Molecular Cloning: A
Laboratory Manual Cold Spring Harbor Laboratory Press; 3rd edition
(2001).

[0068] It will be understood that more than one enzyme may be responsible
for a cell death-stable enzyme activity. For example, a group of related
enzymes may share a substrate. In some embodiments, an enzyme activity is
catalyzed by at least 1, 2, 3, 4, 5, 10, 20, or more, different enzymes.

Detection Methods

[0069] A cell death-stable protein can be detected by conventional
techniques known in the art, e.g., Western blot, ELISA, mass
spectrometry, chromatography, or immunochemistry. Alternatively, a cell
death-stable protein can be detected by a characteristic cell
death-stable enzyme activity. That is, a protein may be detected
indirectly by its function, e.g., a reaction which it catalyzes.
Disrupting membrane integrity permits detection of enzyme activity
previously inaccessible to molecules in the extracellular milieu by,
e.g., diffusion of the enzyme out of the cell, entry of a substrate into
the cell, or both.

[0070] The skilled artisan will recognize that combinations of substrates
and leaving groups can be screened for use in the methods of the
invention, without necessarily knowing the enzyme(s) responsible for
catalyzing the release of the leaving group. For example, test samples
may be made from known quantities of viable and non-viable cells (see,
e.g., Example 1) and incubated with a candidate substrate according to
the methods of the invention. The leaving group is then detected and its
intensity plotted against the known ratio of viable and non-viable cells.
Useful substrates will be those that bear a linear correlation with the
known proportion of viable and non-viable cells. By testing substrates in
this way, it is not necessary to know the source(s) of the cell
death-stable enzyme activity.

[0071] Enzyme substrate/leaving group conjugates are well known in the
art. A useful property of such compounds is the internal quenching of the
detectable leaving group. That is, the leaving group is not at all, or
only poorly, detectable when conjugated to an enzyme substrate, but
rapidly becomes detectable upon dissociation from the substrate, e.g.,
following enzymatic processing of the enzyme substrate. Classes of
leaving groups that may be used in the methods of the invention include,
but are not limited to, chromogenic, fluorescent, and luminescent
molecules.

[0072] Chromogenic molecules for the detection of enzyme activity are well
known in the art. Tetrazolium salts and formazans were some of the first
substrates used to detect enzymatic activity (Altman, Prog. Histochem.
Cytochem., 9:1-56 (1976)). Additional colorimetric compounds may be found
in, e.g., U.S. Pat. No. 7,026,111, at column 11.

[0073] Luminescent molecules, such as luminol and isoluminol, can be
conjugated to enzyme substrates and used directly in the methods of the
invention (see, e.g., U.S. Pat. No. 4,748,116). Alternatively, substrates
conjugated to luciferin can be employed in a system where luciferase is
expressed (see, e.g., U.S. Pat. No. 7,148,030).

[0075] The signal produced by the leaving group may be detected by any
appropriate means, e.g., visual inspection, a spectrophotometer,
luminometer, or fluorometer. In applications where two or more
distinguishable leaving groups are present in a sample, they may be
detected simultaneously or sequentially.

[0076] Substrate-leaving group conjugates useful in the methods of the
invention will have a leaving group conjugated to a substrate of a cell
death-stable enzyme that is, e.g., a carbohydrate, lipid, protein,
peptide, nucleic acid, hormone, or vitamin moiety; or a combination of
one or more such substrates. These moieties may be naturally-occurring
(e.g., biochemically purified) or synthetic (e.g., chemically synthesized
or recombinantly produced). Additionally, these substrates may contain
no, some, or all non-native components (e.g. non-natural amino acids,
blocking or protecting groups, etc.). Extensive catalogs of
enzyme/substrate pairs are known in the art (see, e.g., U.S. Pat. Nos.
4,167,449 (particularly Table II), 5,871,946 (particularly Table I), and
7,026,111 (particularly columns 13-18) for examples of such
enzyme/substrate pairs). Additionally, substrate libraries may be
generated, as disclosed in U.S. Pat. No. 6,680,178, and screened to
identify useful peptide substrates for use in the methods of the
invention. In some embodiments, an enzyme activity's substrate Preference
can be profiled using phage display technology, as disclosed in, e.g.,
Felber at al., Biol. Chem. 386:291-98 (2005).

[0078] The central role of proteases in maintaining cellular and
organismal homeostasis across phyla is one reason for the prevalence of
labeled peptide substrates as markers of protease activity (see, e.g.,
U.S. Pat. Nos. 6,037,137 and 6,984,718, which provide reagents and
methods for detecting protease activity in situ and in whole cells).

[0079] Intrinsic enzyme activity varies widely among different enzymes and
for different substrates of a particular enzyme. Extrinsic factors
affecting enzyme activity include the conditions of the medium (e.g., pH,
temperature, osmolarity, etc.), the expression level or
post-translational regulation of the enzyme, and substrate concentration.
Substrate concentration will need to be adjusted by the practitioner
appropriately. For a given enzyme, and medium conditions, suitable
substrate concentrations may be in the range of, e.g., 0.01 ng/ml to 100
mg/ml, or 10 μg/ml to 10 mg/ml. In some situations, a substrate
concentration of between 0.001 mM and 10 mM may be appropriate.
Alternatively, the substrate concentration can be between 0.01 mM and 0.5
mM. Similarly, incubation times that allow for the development of
detectable signals, will vary widely depending on these same parameters.
Accordingly, incubation times may range from 30 seconds or less, up to 1,
2, 3, 5, 10, 20, 30, 45, 60, 75, or 90 minutes; or even 2, 4, 6, 10, or
12 hours, or more.

[0080] One useful substrate for the detection of proteolytic activity in
the methods of the invention is bis-(Ala-Ala-Phe)-Rhodamine-110 (Promega,
Cat. No. G9260). An additional substrate useful in the methods of the
invention is Ala-Ala-Phe-AMC (Sachem Cat No. 1-1415.0050). It is
theorized, but not relied upon, that the Ala-Ala-Phe tripeptide is a
substrate for the extralysosomal tripeptidyl peptidase II enzyme (TPP II;
Balow et al., J. Biol. Chem., 261:2409-2417 (1986)) and the lysosomal
tripeptidyl peptidase I enzyme (TPP I; Vines and Warburton, Biochim.
Biophys. Acta., 1384:233-242 (1998) and Steinfeld et al., J. Histochem.
Cytochem., 54:991-996 (2006)). Notably, Ala-Ala-Phe is a common and
specific substrate for the bacterial subtilisins (Stambolieva et al.,
Arch. Biochem. Biophys., 294:703-6 (1992)), which are functionally
similar to the tripeptidyl peptidases. Additional substrates of TPP I may
be found in, e.g., Tian et al., J. Biol. Chem., 281:6559-72 (2006), which
screened large libraries of substrates and U.S. Pat. No. 6,824,998, which
disclosed substrates (with precipitating leaving groups) useful for
histological applications.

[0081] Ala-Ala-Phe is known to also be a substrate for the chymotrypsin
enzyme. Other substrates for chymotrypsin and related enzymes, such as
calpain, are known in the art--as are structure/function correlations of
such enzymes. These are discussed further in, e.g., Sharma et al., Biol.
Chem. (2008; Aug. 8 electronic publication; PubMed Id (PMID) No.
18690777), Croall and Ersfeld Genome Biol. 8:218 (2007); Czapinska and
Otlewski Euro. J. Biochem 260:571-95 (1999); Perona and Craik J. Biol.
Chem. 272:29987-90 (1997).

Cell Culture Medium, and Matrix

[0082] The invention provides methods which may be used to measure the
viability of cultured cells derived from a wide variety of host
organisms, e.g., mammals, including humans, and from a wide variety of
source tissues. The cells assayed may be derived from tissues in various
stages of development. Cells may be derived from an adult, fetal, or
embryonic source. The cells may be totipotent or pluripotent stem cells,
derived from an organ originating from any of the three primordial germ
layers (i.e., ectoderm, mesoderm or endoderm). For example, cells may be
derived from skin, heart, skeletal muscle, smooth muscle, kidney, liver,
lungs, bone, pancreas, central nervous tissue, peripheral nervous tissue,
circulatory tissue, lymphoid tissue, intestine, spleen, thyroid,
connective tissue (e.g., chondrocytes), or gonad. The cells may be
non-expanded primary cells, culture-expanded primary cells, or
established cell lines. Additionally, the cells may be grown in a variety
of media, e.g., with or without serum (e.g., chemically defined media),
and with or without phenol red.

[0083] The invention provides methods to measure the viability of cells
over a wide range of cell densities. For example, the cells may be
present at a density of between 2.2×104 and 2.8×106
cells/cm2, between 3.5×104 and 2.8×106, or
between 5×104 and 1×106 cells/cm2. The cells
may also be present at a high density of at least 2.0×105,
5.0×105, 1.0×106, 2.0×106,
2.8×106, 3×106, 4×106, 5×106,
6×106, 8×106, 10×106 cells/cm2, or
more. The methods provided by the invention have been practiced with cell
densities of up to about 3×106 cells/cm2. It is
contemplated that the methods would work with cell densities of up
106 cells/cm2, or more. It should be understood that all cell
densities referenced throughout this disclosure are qualified by the term
"average." The skilled artisan will undoubtedly appreciate that local
fluctuations in cell density will occur and are contemplated in the
methods provided by the invention.

[0084] Cells are incubated in medium to allow for the accumulation of cell
death-stable protein or enzyme activity in the medium, i.e., to produce
conditioned medium. The methods provided by the invention measure
viability over the amount of time that the cells are in contact with the
medium, i.e., conditioned medium generally cannot be replaced with fresh
medium just before assay. Cells may be incubated for a variable amount of
time, depending on the particular application, e.g., cell type, cell
density, medium type, or half-life of the cell death-stable protein or
enzyme activity. Cells may be incubated before assaying for about 1, 5,
10, 30, 60, 90, 120, 150, 180, 210, or 240 minutes; or about 3, 4, 5, 6,
8, 10, 12, 18, or 24 hours; or up to about 1, 2, 3, 4, 5 days, or more.

[0085] The methods of the invention are useful to measure the fraction of
viable cells grown on a variety of substrates or matrices. Cells may be
grown on traditional two-dimensional cell culture substrates, e.g., glass
or surface treated plastic. Alternatively, cells can be supported by a
scaffold or matrix, e.g., where the cells are part of a tissue engineered
product. Suitable scaffolds may include structures composed of metals,
plastics, glass, silicon, ceramics, and/or calcium phosphates. Other
suitable scaffold materials include absorbable polyesters (e.g. polymers
of glycolide or lactide, and derivatives or copolymers thereof);
carbohydrate (e.g. hyaluronin, chitin, starch, or alginate); and protein
(e.g., collagen (e.g., a porcine collagen-derived matrix) or gelatin), or
combinations of any of these matrices. Further discussion of matrices
used in tissue engineering may be found in, e.g., Langer and Vacanti
(1993); Ikada, J. R. Soc. Interface, 3:589-601 (2006); and U.S. Pat. Nos.
6,689,608 and 6,800,296.

Assay Variations

[0086] Applicants have discovered that phenol red can further extend the
range of cell densities assayable by the methods of the invention. This
is achieved by attenuating the signal of the leaving group. That is,
phenol red reduces the signal of, e.g., rhodamine-110 (R110), and the
assay saturates at a higher cell density. It is theorized, but not relied
upon, that deprotonated phenol red exerts this effect because its
absorption spectrum has significant overlap with both the excitation and
emission spectra of rhodamine 110.

[0087] In addition to phenol red for R110, the use of other attenuating
agents adapted for use with other leaving groups is also contemplated.
Appropriate attenuating agents for particular leaving groups' excitation
and or emission spectra will have the desired degree of overlap in its
absorption spectrum. Absorption, excitation, and emission spectra are
known in the art or may be readily determined empirically, e.g., by
fluorometry.

[0088] Furthermore, the methods provided by the invention are modular and
amenable to multiplexing. That is, additional processes, steps, and/or
agents can further extend an assay's utility. For example, the methods
provided by the invention may further include the detection of more than
one cell death-stable protein or enzyme activity. This is achieved by
applying multiple enzyme-specific substrates for two or more cell
death-stable enzyme activities in a sample portion using, e.g.,
orthogonal substrates and/or leaving groups. Such a "detection mixture"
contains one or more species of substrate for cell death-stable enzyme
activities, coupled to one or more detectable leaving groups. These
multiplexing methods can be divided into two broad classes: a single
species of leaving group, and multiple species of leaving groups.

[0089] A detection mixture where a single species of leaving group is
coupled with multiple species of enzyme substrates will produce an
integrated signal. That is, the resulting signal is a sum of the detected
enzyme activities. For example, each substrate could be processed by a
distinct enzyme activity. By assaying and summing over multiple enzyme
activities, the integrated signal is a more accurate view of the sample's
overall metabolic state. Integrated signals may also be useful where
shorter incubation times are desired.

[0090] The use of multiple species of leaving groups in the methods of the
invention provides independent measures of viability. A detection mixture
containing a single species of substrate for an enzyme activity coupled
to multiple species of leaving groups provides parallel measures of
viability. The different signals offer additional flexibility to
investigators using detection equipment which may have machine or
detector dependent sensitivities, e.g., at different wavelengths and or
intensities.

[0091] The use of multiple substrate species, each coupled to a different
species of leaving group, offer fully independent measures of viability.
The substrates may, e.g., belong to enzymes with low, medium, or high
relative activities. The relative activities could vary from low to high
by at least 10, 20, 40, or 80%, or by at least 1, 2, 5, 10, 50, 100, 500,
or 1000 fold, or more. By making multiple independent measures of
viability, an investigator may be more likely to remain in the linear
detection range with at least one substrate species.

[0092] A further application using multiple leaving groups is a quality
control assay. In particular, the methods of the invention may further
include the step of adding one or more contaminant-specific substrates,
each coupled to the same species of leaving group, and detecting one or
more contaminant-specific enzyme activities. The contaminant-specific
substrate species are substrates for enzymes specific to common cell
culture contaminants such as: fungi, bacteria, archaea, and protists--and
absent from the cultured cells' proteomes. Accordingly, detection of the
contaminant-specific leaving group indicates contamination of the
cultured cell population. Naturally, the leaving group for the
contaminant-specific enzyme activities will be distinguishable from the
leaving group(s) used to measure the viability of the cultured cells.

[0093] The methods of the invention can also be adapted to measure the
cytotoxicity of a treatment. A treatment may be an environmental or
physiological treatment, e.g., thermal, barometric, mechanical, or photic
stimulus. Treatment may also be a chemical treatment, e.g., osmolarity,
pH, a pharmacological or biological agent, or any combination of the
above. The methods of the invention may further include the steps of
applying a treatment to a test cell population, measuring the viability
of the test cell population by the methods of the invention and comparing
the viability to a control culture of the same cells not exposed to the
treatment. In certain embodiments, the cytotoxicity may be calculated as
1 minus the fractional viability of a population. In these embodiments, a
control population is not necessary.

EXAMPLES

Example 1

Measuring Cell Viability of a Tissue-Engineered Product

[0094] A brief schematic of a cell viability assay is shown in FIG. 1.
There, "Read #1" is the amount of cell death-stable or enzyme activity
present in the portion of the population containing the cells and
conditioned medium, while "Read #2" is the amount of cell death-stable
protein of enzyme activity present in the portion of the conditioned
medium not containing cells of the culture population.

[0095] Human articular chondrocytes were expanded to second or third
passage in monolayer cultures. In order to replicate culture conditions
used in MACI® implants, chondrocytes were seeded in triplicate onto
white opaque 96 well plates on the rough side of ACI-MAIX® membrane
matrix punches (6 mm in diameter) at densities of approximately 25,000 to
600,000 cells per punch. Matricel ACI-MAIX® membrane matrix is a
porcine collagen based membrane matrix with a smooth side and a rough
side. This seeding density is equivalent to 8.75×104 to
2.1×106 cells/cm2 which corresponds to
1.75×106 to 42×106 cellsper ACI-MAIX® membrane
matrix (20 cm2). When the assay is applied to full-sized MACI®
implant samples, two small punches (typically 6 mm in diameter) and a
proportional amount of conditioned medium are taken from each sample. For
two punches 6 mm in diameter, which together represent approximately 2.8%
of a 20 cm2 membrane, a proportional amount of the conditioned
medium is approximately 2.8% of the total volume of the conditioned
medium overlying the 20 cm2 membrane. In both the full-scale and
downscaled cases, blank membrane matrix punches and medium were processed
as controls.

[0096] Three hours after cell seeding, half of the conditioned medium,
which would contain half of the total amount of any proteases released by
dead (nonviable) cells, was transferred to empty wells.

[0101] After incubation (45-90 min.), the plate was read using a Molecular
Devices SpectraMax M5 Microplate Reader with the SoftMax Pro Software at
excitation 485 nm-emission 520 nm. The data was then processed in
Microsoft EXCEL.

[0102] Results of representative experiments are shown in FIG. 2.
Scatterplots of fluorescent reporter signal strength, (Read #1, live
cells with supernatant, FIG. 2A; and Read #2, supernatant only, FIG. 2B);
as a function of cell seed density are shown. Data points are the average
of three replicates. Incubation time with the
bis-(Ala-Ala-Phe)-Rhodamine-110 substrate was either 45, 60, or 90
minutes. The relationship between signal and cell density was linear and
varied little for all incubation times tested. A 60 minute incubation
step was used in subsequent measurements.

Example 2

Assay Accuracy and Precision

[0103] The accuracy of the assay was evaluated by comparing the measured
viability of a culture with a known percentage of viable cells. The
culture was composed of a mixture of known quantities of live and dead
cells, pre-mixed and seeded at the indicated densities, then processed as
in Example 1. The measured viability was plotted as a function of the
percent of viable cells in the test mixture (FIG. 3). The plotted data
points are the average of two replicates. Typically the difference
between the measured viability and the actual viability is less than 15%.
For cell seeding densities lower than 0.175×106
cells/cm2, a longer incubation time (at least 90 min.) helps ensure
assay accuracy.

[0104] To measure the inter-strain accuracy of the assay, 1:1 proportions
of live and dead cells from 3 different strains were seeded at a density
of 7.0×106 cells/cm2. Although significant intrinsic
variability can exist in the absolute signal levels from different cell
strains (Table 1, first data column; % CV=41.49) the variability in
measured viability is substantially less (Table 1, second data column; %
CV=7.62).

Contribution of Matrix, Reagent, and Analyst Variability to Assay
Precision

[0105] In order to assess the effect of different analysts and different
matrix or reagent lots on the precision of measured viability, cells from
a single parent culture were seeded at a density of 7.0×105
cells/cm2 on membrane punches and processed as described in Example
1. Three variables were analyzed: matrix lot, assay lot, and analyst.
Each variable was tested in two groups--each treatment group having three
statistical replicates. The results are shown in Table 2.

These results suggest that the assay is relatively insensitive to changes
in these technical variables.

[0106] During the development of this assay, it was found that the
addition of phenol red to the assay mixture could attenuate the signal
intensity in a dose-dependent manner, and extend the linear range of the
assay to higher cell densities. Cells were seeded at varying densities
and processed as in Example 1, with or without phenol red and the average
viability of three replicates is shown in FIG. 4. The addition of phenol
red does not affect the accuracy of the assay, it merely serves to
prevent the signal levels from approaching saturation by suppressing the
signal outputs in a dose-dependent manner. The amount of phenol red can
be adjusted as needed. Phenol red is not typically needed for seeding
densities lower than 0.5×106 cells/cm2 membrane matrix.

Example 5

Timing of Phenol Red Addition

[0107] The timing of addition of phenol red to the assay mixture was found
to be flexible. To demonstrate this, cells from a single strain were
sonicated to release all intracellular proteases and seeded at a density
of 1.0×104 cells/well in a 96 well plate, in a volume of 100
μl/well. Substrates and phenol red were added in the amount and at the
time according to Table 3. The results are shown as the average signal
intensity of three replicates per treatment in Table 4. These results
demonstrate that phenol red of varying concentrations added at varying
time points during the assay is similarly effective in attenuating the
signals.

[0108] The use of phenol red significantly expanded the dynamic range of
the new assay to measure viability when using either: a high cell seeding
density (typically over 1.4×106 cells/cm2), or in a
variety of media including serum containing or serum free, and phenol red
containing or phenol-red free.

[0109] To demonstrate the effect of a different matrix material on the
method, cells were seeded on Gelfoam (Upjohn Pharmacia, Kalamazoo,
Mich.), a highly porous gelatin sponge, at densities ranging from
7.1×104 to 2.3×106 cells/cm2. Cells were
processed as in Example 1. Results are shown in FIG. 5 as the average
viability of three replicates. The viability of the cells, as determined
by trypan blue exclusion just prior to seeding, was 91%. These data
indicate that the assay is amenable to analyzing cells seeded on a
variety of different matrices.

Example 7

Cells Grown on 2D Cultures (Tissue Culture Plastic)

[0110] To demonstrate the effectiveness of the assay in a more traditional
tissue culture environment (i.e. growth on an inorganic, flat substrate),
cells were seeded directly in a plastic six well tissue culture plate,
with no matrix, at densities ranging from 2.2×104 to
2.8×106 cells/cm2. The cells were processed as in Example
1. The results are shown in FIG. 6 as the average viability of two
replicates. The viability of the cells, as determined by trypan blue
exclusion just prior to seeding, was 96%. These results demonstrate that
the assay performs well with cells grown on a traditional cell culture
substrate, in addition to cells grown on a variety of matrices.

Example 8

Non-Human Cells

[0111] To demonstrate the effectiveness of the assay on non-human cells,
rabbit chondrocytes, from two donors, were seeded on ACI-MAIX®
membrane matrix punches (6 mm in diameter) at densities ranging from
0.175 to 1.4×106 cells/cm2. The cells were processed as
in Example 1. The results are shown in FIG. 7 as the average viability of
two replicates. The viability of strains 1 and 2, as determined by trypan
blue exclusion just prior to seeding, were 88.0% and 84.9%, respectively.
These results demonstrate that the assay performs well with cells from a
non-human source.

Example 9

Alternative Substrate

[0112] To demonstrate the effectiveness of the method using substrates
other than the bis-(Ala-Ala-Phe)-Rhodamine-110, an alternative substrate,
(Ala-Ala-Phe)-AMC (Sachem Cat No. 1-1415.0050, Torrance, Calif.), was
tested using three strains of human chondrocyte seeding at densities in
the range of 8.75×104 to 1.4×106 cells/cm2.
The cells were processed as in Example 1, except the
bis-(Ala-AlaPhe)-Rhodamine-110 was replaced by (Ala-Ala-Phe)-AMC and the
sample plate was read at excitation 360 nm-emission 440 nm. The
viabilities for stain A, B, and C, determined by trypan blue exclusion
prior to seeding, were 98.6%, 98.6%, and 99.2%, respectively. The results
are shown in FIG. 8 as the average viability of two replicates. The
result demonstrated that the alternative (Ala-Ala-Phe)-AMC substrate was
effective (FIG. 8).

[0113] For all patent, application, or other reference cited herein, it
should be understood that it is incorporated by reference in its entirety
for all purposes as well as for the proposition that is recited. Where
any conflict exits between a document incorporated by reference and the
present application, this application will dominate.

[0114] Other embodiments of the invention will be apparent to those
skilled in the art from consideration of the specification and practice
of the invention disclosed herein. It is intended that the specification
and examples be considered as exemplary only, with a true scope and
spirit of the invention being indicated by the following claims.